Cloning and expression of the MspI restriction and modification genes

The genes for the MspI restriction (R) and modification enzymes (recognition sequence CCGG) have been cloned into Escherichia coli using the vector pBR322. Clones carrying both genes have been isolated from libraries prepared with EcoRI, HindIII and BamHI. The smallest fragment that encodes both activities is a 3.6-kb HindIII fragment. Plasmids purified from the clones are fully resistant to digestion by MspI, indicating that the modification gene is functional in E. coli. The clones remain sensitive to phage infection, however, indicating that the endonuclease is dysfunctional. When the R gene is brought under the control of the inducible leftward promoter from phage lambda, the level of endonuclease increases and the level of methylase decreases, suggesting that the genes are transcribed in opposite directions.     

Overproduction of the EcoR V endonuclease and methylase.

Strains overproducing the EcoR V endonuclease and methylase have been obtained by inserting each of the two genes in expression vectors containing the lambda PL promoter. The methylase is overproduced to a level reaching 5-10% of the total cellular proteins, which represents a 50-100 fold increase. A 30 fold overproduction of endonuclease was achieved by randomly positioning the EndRV gene downstream of the lambda PL promoter. The situation in the endonuclease overproducing clone resembles that encountered in maxi-cells. The strains described here allowed a quick purification of both enzymes in sufficient amounts for crystallisation attempts.     

Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism.

Two novel types of alleviation of DNA restriction by the EcoKI restriction endonuclease are described. The first type depends on the presence of the gam gene product (Gam protein) of bacteriophage lambda. The efficiency of plating of unmodified phage lambda is greatly increased when the restricting Escherichia coli K-12 host carries a gam+ plasmid. The effect is particularly striking in wild-type strains and, to a lesser extent, in the presence of sbcC and recA mutations. In all cases, Gam-dependent alleviation of restriction requires active recBCD genes of the host and recombination (red) genes of the infecting phage. The enhanced capacity of Gam-expressing cells to repair DNA strand breaks might account for this phenomenon. The second type is caused by the presence of a plasmid in a restricting host lacking RecBCD enzyme. Commonly used plasmids such as the cloning vector pACYC184 can produce such an effect in strains carrying recB single mutations or in recBC sbcBC strains. Plasmid-mediated restriction alleviation in recBC sbcBC strains is independent of the host RecF, RecJ, and RecA proteins and phage recombination functions. The presence of plasmids can also relieve restriction in recD strains. This effect depends, however, on the RecA function in the host. The molecular mechanism of the plasmid-mediated restriction alleviation remains unclear.     

Properties of the replicator region of the natural plasmid pLG13 containing genes of the EcoRV restriction-modification system]

The previously constructed plasmid pILRV8 that induces endonuclease EcoRV gene overexpression kills cells of some E. coli strains under the induction of this enzyme synthesis. Cell transformation by natural plasmid pLG13 carrying genes of the EcoRV restriction--modification system was found to appreciably enhance cell viability ("survival") under endonuclease overproduction. A plasmid pLG13 region located in immediate proximity to the methylase gene was shown to be responsible for the above effect. This region was also capable for autonomous replication. The analysis of the DNA primary structure in the found replicator region allowed to refer the pLG13 to ColE1 family plasmids. Perturbations in the region lead to loss of the "survival" effect and change of the plasmid replicative properties. A relationship between the replicon elements, the EcoRV genes region and "survival" effect is discussed. Based on the replicon found multicopy vector molecules have been constructed.     

Alleviation of type I restriction in the presence of plasmids of group inc I. General characteristics and molecular cloning of the ard gene

The capability of a number of plasmids of incN and incI groups to alleviate an action of type I EcoK, EcoB, EcoD, and EcoA restriction endonucleases on the unmodified DNA was revealed. The efficiency of EcoK action on lambda 0 DNA is alleviated about 10 divided by 100 fold in E. coli K12 AB 1157 bacteria containing the plasmid of incN group (pKM101, N3, pJA4733) or incI group (R144, R648; R621a; ColIb-P9). We have cloned ard gene of ColIb-P9 plasmid (SalI-C fragment) in pBR322 multicopying vector. A hybrid clone abolishing the EcoK restriction has been received. Ard gene activity is independent of the recA, recBc, recF, lexA, umuC, lon bacterial genes activity. Ard gene's product does not inhibit the EcoK restriction endonuclease action as well as ocr protein (phage T7) and does not increase the process of methylation of DNA as well as ral protein of phage lambda.     

High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda

Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using lambda pL promoter expression vectors. In a first step we constructed endRI::lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing EcoRI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the overproduced EcoRI endonuclease.     

Transactivation of p'R promoter of phage lambda

The host-vector system for efficient expression of the cloned genes under the control of transactivated promoter p'R of bacteriophage lambda has been elaborated. The Q protein activating p'R promoter is coded by the defective prophage constructed in vitro by means of excision of the late phage genes between the distant sites of the restriction endonuclease MluI and change of the central SalI fragment carrying the kill gene for the kanamycin resistance gene. The general recombination system is impaired during the change, thus the bacteriophage DNA can be obtained from the induced RecA cells as a plasmid DNA. The induction of the prophage results in a sharp increase of beta-lactamase synthesis (30% of soluble cell protein) under the control of p'R promoter in a plasmid derived of pBR322.     

Various characteristics of the anti-restriction mechanism in bacteriophage T5

Bacteriophage T5 is not confined by the restriction systems of the second type EcoRII and EcoRV. Bacteriophage T5 DNA is not modified by EcoRII and EcoRV methylases in vivo. The sites of recognition for restriction endonuclease EcoRV are mapped at 24.4; 57.6; 68.5; 70.2% of T5 DNA, while the sites at 5.1; 7.6% are recognized by EcoRII, the sites at 5.75; 6.0 and 6.5% are recognized by HpaI in FST. A high activity of restriction endonucleases EcoRI and EcoRV is demonstrated in crude extracts of E. coli B834 (RI) and E. coli B834 (RV) cells infected by bacteriophage T5. The simultaneous infection of E. coli B834 (RI) or E. coli B834 (RV) cells by the amber mutants of bacteriophage T5 and the suppressing phage lambda NM761 does not result in the protection of lambda DNA by the T5 anti-restriction mechanism. The presented data support the hypothesis that the anti-restriction mechanism of bacteriophage T5 is based on prevention of T5 DNA contacts with restriction enzymes by a specific phage protein.     

Purification of the HhaII restriction endonuclease from an overproducer Escherichia coli clone

An Escherichia coli K12 strain carrying the HhaII methylase and restriction genes on two separate compatible plasmids, pSK5 and pSK7, is used to overproduce the restriction endonuclease. Plasmid pSK5 expresses the methylase gene constitutively from its chloramphenicol resistance gene promoter, and plasmid pSK7 expresses the restriction endonuclease under control of the lacUV5 promoter. Induction of the two-plasmid clone with 1 mM isopropyl-1-thio-beta-D-galactopyranoside results in a 15-fold increase in HhaII endonuclease activity. The enzyme has been purified to apparent homogeneity. It migrates as a 23-kilodalton polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide electrophoretic gels and as a 52-kilo-dalton native protein dimer on a high pressure liquid chromatography sizing column.     

Genetic complementation by cloned bacteriophage T4 late genes.

Bacteriophage T4 containing nonsense mutations in late genes was found to be genetically complemented by four conjugate T4 genes (7, 11, 23, or 24) located on plasmid or phage vectors. Complementation was at a very low level unless the infecting phage carried a denB mutation (which abolishes T4 DNA endonuclease IV activity). In most experiments, the infecting phage also had a denA mutation, which abolishes T4 DNA endonuclease II activity. Mutations in the alc/unf gene (which allow dCMP-containing T4 late genes to be expressed) further increased complementation efficiency. Most of the alc/unf mutant phage strains used for these experiments were constructed to incorporate a gene 56 mutation, which blocks dCTP breakdown and allows replication to generate dCMP-containing T4 DNA. Effects of the alc/unf:56 mutant combination on complementation efficiency varied among the different T4 late genes. Despite regions of homology, ranging from 2 to 14 kilobase pairs, between cloned T4 genes and infecting genomes, the rate of formation of recombinants after T4 den:alc phage infection was generally low (higher for two mutants in gene 23, lower for mutants in gene 7 and 11). More significantly, when gene 23 complementation had to be preceded by recombination, the complementation efficiency was drastically reduced. We conclude that high complementation efficiency of cloned T4 late genes need not depend on prior complete breakage-reunion events which transpose those genes from the resident plasmid to a late promoter on the infecting T4 genome. The presence of the intact gene 23 on plasmids reduced the yield of T4 phage. The magnitude of this negative complementation effect varied in different plasmids; in the extreme case (plasmid pLA3), an almost 10-fold reduction of yield was observed. The cells can thus be said to have been made partly nonpermissive for this lytic virus by incorporating a part of the viral genome.     

Synthesis of a trans-Acting Inhibitor of DNA Maturation by Prohead Mutants of Phage λ

Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.     

lambda-Transducing phages carrying transfer genes isolated from an abnormal prophage insertion into the traY gene of F

A set of lambda-transducing phages carrying transfer (tra) genes has been isolated from an abnormal lysogen in which a lambda prophage was inserted into the traY gene of Flac. These have been characterized genetically for complementation of Flac tra and finP point mutants and for the presence of oriT. Studies of tra gene expression during lambda repression showed that tra genes on the transducing phages were expressed from the lambda PL promoter as well as from the transfer promoters when these were present. The molecular weights of the traM (14,000) and traJ (23,500) proteins were measured after infection of ultraviolet-irradiated cells with one of the phages, ED lambda 102, and overproduction of the traJ protein upon induction of an ED lambda 102 lysogen was demonstrated. A proportion of this traJ protein was located in the inner membrane and cytoplasmic fractions of the cell, the majority being in the outer membrane. Physical analysis of the DNA carried by the lambda tra phages by determination of the phage buoyant densities in CsCl, by restriction enzyme digestion and by electron microscope heteroduplex analysis, was used to define the DNA segments encoding the tra functions. Correlation of the physical and genetical data improved the positioning of the tra genes within the transfer region. These results were combined with new restriction enzyme cleavage data to construct an improved map of this region.     

Cloning, expression, and purification of gene 3 endonuclease from bacteriophage T7

The structural gene for the single-stranded endonuclease coded for by gene 3 of bacteriophage T7 has been cloned in pGW7, a derivative of the plasmid pBR322, which contains the lambda PL promoter and the gene for the temperature-sensitive lambda repressor, cI857. The complete gene 3 DNA sequence has been placed downstream of the PL promoter, and the endonuclease is overproduced by temperature induction at mid-log phase of Escherichia coli carrying the recombinant plasmid pTP2. Despite the fact that cell growth rapidly declines due to toxic effects of the excess endonuclease, significant amounts of the enzyme can be isolated in nearly homogeneous form from the induced cells. An assay of nuclease activity has been devised using gel electrophoresis of the product DNA fragments from DNA substrates. These assays show the enzyme to have an absolute requirement for Mg(II) (10 mM), a broad pH optimum near pH 7, but significant activity from pH 3 to pH 9, and a 10-100-fold preference for single-stranded DNA (ssDNA). The enzyme is readily inactivated by ethylenediaminetetraacetic acid or high salt. The differential activity in favor of ssDNA can be exploited to map small single-stranded regions in double-stranded DNAs as shown by cleavage of the melted region of an open complex of T7 RNA polymerase and its promoter.